Method and apparatus for manufacturing beads array chip

ABSTRACT

Provided is a convenient process of manufacturing a two-dimensional bead array on a chip by disposing a plurality of beads immobilized with a biomolecular probe in a bead array container in a predetermined order. The bead array chip is manufactured by repeating the following operations: a container having a plurality of first channels disposed in parallel with each other and a second channel crossing therewith is retained in a container retaining portion and a capillary is moved through the second channel of the container vertically. The capillary is moved downward to suck and retain, in an end thereof, one bead stored in a storing portion of a bead storing plate and then, moved upward to the position of a desired first channel. Under this state, pure water is fed to the first passage from a water feed system and a water stream is generated by sucking the pure water by a suction pump. The bead retained in the end of the capillary is transferred by the water stream and is then, blocked by a dam disposed in the first channel, in which the bead is retained.

FIELD OF THE INVENTION

[0001] The present invention relates to a manufacturing process andapparatus of a bead array chip which introduces and disposes minutebeads having a surface immobilized with a biomolecular probe one by onein a microfluidic channel formed in a bead array container.

BACKGROUND OF THE INVENTION

[0002] With the progress of the human genome project, a movement tounderstand a living body at the DNA level and utilize the knowledge thusobtained for disease screening or understanding of the life phenomenonhas become active. Investigation of a gene expression condition iseffective for understanding of the life phenomenon or genetic activity.As a powerful method for finding the gene expression condition, aso-called DNA chip, that is, a probe array having a number of DNA probesdivided by kind and immobilized on a surface of a solid such as slideglass has recently been employed. Examples of a manufacturing method ofa DNA chip include a method of synthesizing base oligomers having adesigned sequence one by one in a plurality of divided cells by usingphotochemical reaction and lithography widely employed in asemiconductor industry (Science, 251, 767-773(1991)) and a method ofembedding plural kinds of DNA probes in sections, one probe in onesection (Science, 270, 467-470(1995); Nat. Biotechnol. 18,438-441(2000)).

[0003] Manufacture of a DNA chip by either one of the above-methodstakes labor and time, leading to a high cost, because DNA probes must beimmobilized onto respective arrays or base oligmers must be synthesizedone by one in each array. These methods are accompanied with thedrawbacks that the density of the probes differs with the sectionbecause they are disposed as liquid drops on the surface of a solid,combination of probe kinds cannot be easily changed, and manipulation ofthem is difficult for users.

[0004] With a view to overcoming the above-described problems, probearray having plural kinds of beads each having a DNA probe fixedthereon, that is, a bead array (Clinical Chemistry, 43, 1749-1756(1997);Science 287, 451-452(2000); Nucleic Acids Research 30, e87(2002)) wereproposed. When beads are used, a probe array free from variations inprobe density by bead can be manufactured, because a chemical reactionin a solution can be utilized for the immobilization of the probe.

[0005] In a DNA chip, the kind of a probe is discriminated by theposition where an oligomer is prepared or spotting position of each DNAprobe or protein probe, while in the probe array using probe-immobilizedbeads, it is discriminated by using beads different in color (ClinicalChemistry, 43, 1749-1756(1997); Science, 287, 451-452(2000)) or by thearraying order of beads in the capillary (Nucleic Acids Research 30,e87(2002)).

[0006] In a DNA chip, a sample to be analyzed is reacted with anoligomer or DNA immobilized onto the DNA chip while spending a half aday or all day for identification and quantitative analysis of pluralkinds of DNAs contained in the sample. In a probe array having beadsarranged in a capillary, that is, a bead array, on the other hand, asample to be analyzed is forced to flow in a capillary. Bead array needsless time for genetic testing than the conventional method so that it isa measuring technique suited for use in medical sites such as hospitals.For example, in an infection requiring urgent diagnosis or a bacterialtest, it is expected as rapid detecting means of an exogenous gene of apathogenic microorganism genome not existing in the genome of thepatient.

[0007] For industrialization of a bead array, it is essential toestablish a method capable of selecting desired probe-immobilized beads,depending on the purpose of the test, and arraying them freely. A methodof pouring beads, under control, in a capillary one by one by making useof the flow of a liquid (Japanese Patent Application Laid-Open No. Hei11-243997); and a method of retaining one bead, among a plurality ofbeads introduced together with a solvent, in a microhole made in a sheetand permitting insertion of only one bead, transferring the sheet to acapillary or groove formed in a plate, and thus arranging the beads oneby one (Japanese Patent Application Laid-Open No. 2000-346842) wereproposed so far.

[0008] Non-patent Document 1: Science, 251, 767-773(1991)

[0009] Non-patent Document 2: Science, 270, 467-470(1995)

[0010] Non-patent Document 3: Nat. Biotechnol. 18, 438-441(2000)

[0011] Non-patent Document 4: Clinical Chemistry, 43, 1749-1756(1997)

[0012] Non-patent Document 5: Science, 287, 451-452(2000),

[0013] Non-patent Document 6: (Nucleic Acids Research 30, e87(2002)

[0014] Patent Document 1: Japanese Patent Application Laid-Open No. Hei11-243997

[0015] Patent Document 2: Japanese Patent Application Laid-Open No.2000-346842

SUMMARY OF THE INVENTION

[0016] The conventional bead array manufacturing methods however involveproblems in reliability and ease of manipulation, because air bubblesoften disturb smooth introduction of beads in a capillary.

[0017] An object of the present invention is to provide a bead arraymanufacturing apparatus and manufacturing method by introducing andarraying, in a predetermined order, beads having a desired biomolecularprobe such as DNA, RNA or protein immobilized thereon in a bead arraycontainer and forming a two-dimensional bead array on one chip in aconvenient manner.

[0018] In the present invention, a bead capturing capillary capable ofmoving while sucking beads in an end of the capillary is used asmanipulating means for taking out beads from a storing portion of a beadstoring plate one by one and transferring it to a desired position. Anoptically transparent glass or resin container (bead array container)having a structure in which a bead disposing channel wide enough topermit passage of only one bead and a capillary passage wide enough topermit passage of the bead capturing capillary and serving as a guide ofthe bead capturing capillary cross each other is prepared as a containerfor retaining and arraying a plurality of beads in a predeterminedorder.

[0019] The bead capturing capillary has an inner diameter smaller thanthe diameter of the bead and an external diameter greater than thediameter of the bead but not greater than twice as much as the diameterof the bead. The bead capturing capillary is, at one end thereof,connected to a suction pump to generate a suction force at the end ofthe capillary. Members are disposed so that m pieces of bead capturingcapillaries are each inserted from one end of the capillary passage ofeach of m pieces of bead array containers and the end of each capillaryand each storing portion of the bead storing plate keep an oppositepositional relationship therebetween. Both ends of the bead disposingflow channel of the bead array container are linked to a water feedsystem and water suction pump, respectively to cause a water flow in theflow channel.

[0020] A bead is captured from the storing portion of the bead storingplate by projecting the end of the bead capturing capillary from thelower end of the capillary passage of the bead array container, dippingit in the storing portion and then sucking one bead in the end of thecapillary. Then, while the pressure inside of the bead capturingcapillary having one bead retained in one end thereof is kept negative,the capillary is drawn back into the bead array container. When the onebead retained in the end of the capillary comes at the intersect betweenthe capillary passage and bead disposing flow channel, the transfer ofthe bead capturing capillary is stopped and suction in the capillary bythe suction pump is also stopped. At this time, the bead in the end ofthe capillary is still retained in the end by the vacuum conditioninside of the capillary.

[0021] In the next place, pure water is fed from the water feed systemlinked to one end of the bead disposing flow channel, while pure wateris sucked by the water suction pump linked to another end of the beaddisposing flow channel. By making use of the water flow thus generatedin the flow channel, the bead is physically released from the end of thebead capturing capillary to the bead disposing flow channel. The beaddisposing flow channel has, at one end thereof, a dam. The bead thusintroduced remains in the flow channel, blocked by the dam.

[0022] Desired beads can be introduced into the bead disposing flowchannel of the bead array container one by one by the above-describedseries of operations. By specifying an intended position on the beadstoring plate and arraying the desired beads, one by one, in the beaddisposing flow channel of the bead array container in the intendedorder, a bead array chip having a biomolecular probe immobilized thereoncan be manufactured efficiently and inexpensively without failure.

[0023] In one aspect of the present invention, there is thus provided abead array chip manufacturing apparatus, which is equipped with a stagefor supporting a bead storing plate having a plurality of bead storingportions, a stage driving portion for driving the stage, a containerretaining portion for retaining a container which has a plurality offirst (penetrated) channels disposed substantially in parallel with eachother and a second (penetrated) channel crossing with the plurality offirst channels, a capillary which is movable and passes through thesecond channels of the container, a capillary driving portion forvertically moving the capillary, suction means connected to the upperend of the capillary and generates a suction force at the lower end ofthe capillary, fluid circulating means for generating a uniflow of thefluid in the first channels of the container supported at the containerretaining portion, and a controlling portion for controlling the stagedriving portion, capillary driving portion, suction means and fluidcirculating means; and manufactures a bead array chip in which beadsstored respectively in the plurality of storing portions of the beadstoring plate are arranged in a predetermined order in the firstchannels of the container.

[0024] The bead array chip manufacturing process according to thepresent invention is a process for manufacturing a bead array chiphaving plural kinds of bead arrayed in a predetermined order in—in acontainer having a plurality of first channels disposed substantially inparallel with each other and a second channel crossing the firstchannels—each of the first channels, which comprises lowering thecapillary inserted in the second channel and retaining one bead suckedin one end of the capillary; lifting the capillary to position the beadretained in the one end of the capillary in the desired channel amongthe plural first channels; terminating the suction of the capillary; andgenerating a uniflow of a fluid in the first channels.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 is an explanatory view illustrating a bead preparing methodand one structure example of a bead storing plate;

[0026]FIG. 2 is a schematic explanatory view of a container for a beadarray chip;

[0027]FIG. 3 is a schematic view of a bead array chip manufacturingapparatus according to the present invention;

[0028]FIG. 4 is a schematic view illustrating the way how the bead arraycontainer is installed in the bead array chip manufacturing apparatus;

[0029]FIG. 5 illustrates the bead array chip manufacturing apparatus inthe preset mode;

[0030]FIG. 6 illustrates an example of a display screen of a computer;

[0031]FIG. 7A illustrates a step of taking out one bead from the storingportion and introducing it into the bead disposing flow channel;

[0032]FIG. 7B illustrates the step of taking out one bead from thestoring portion and introducing it into the bead disposing flow channel;

[0033]FIG. 7C illustrates the step of taking out one bead from thestoring portion and introducing it into the bead disposing flow channel;

[0034]FIG. 7D illustrates the step of taking out one bead from thestoring portion and introducing it into the bead disposing flow channel;

[0035]FIG. 7E illustrates the step of taking out one bead from thestoring portion and introducing it into the bead disposing flow channel;

[0036]FIG. 7F illustrates the step of taking out one bead from thestoring portion and introducing it into the bead disposing flow channel;

[0037]FIG. 7G illustrates the step of taking out one bead from thestoring portion and introducing it into the bead disposing flow channel;

[0038]FIG. 7H illustrates the step of taking out one bead from thestoring portion and introducing it into the bead disposing flow channel;

[0039]FIG. 7I illustrates the step of taking out one bead from thestoring portion and introducing it into the bead disposing channel;

[0040]FIG. 7J illustrates a washing step of the end of the beadcapturing capillary;

[0041]FIG. 7K illustrates the washing step of the end of the beadcapturing capillary;

[0042]FIG. 8 is a schematic diagram for explaining an image detectionmethod using an image sensor;

[0043]FIG. 9 is a flow chart of a manufacturing process of a bead arraychip;

[0044]FIG. 10 is an explanatory view of a manner of attaching a sampleintroducing jig to a bead array container;

[0045]FIG. 11 is a schematic view illustrating a method of hybridizationtest by using the bead array chip of the present invention; and

[0046]FIG. 12 illustrates the results of the hybridization reactioneffected using the bead array chip of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0047] The embodiments of the present invention will hereinafter bedescribed based on accompanying drawings.

[0048] Referring to FIG. 1, a preparation process of a bead 28 isdescribed. A bead storing plate having m×n storing portions, a pluralityof beads, and a plurality of biomolecular probes such as DNA, RNA orprotein for modifying the bead are prepared. The storing portions 4 onthe bead storing plate 5 are disposed in the direction of x atpredetermined, equally-spaced center distances (first predeterminedcenter distances) and in the direction of y crossing at right angleswith the direction of x at predetermined, equally-spaced distances(second predetermined center distances). The storing portions 4 each hasa columnar shape with an upper circular opening, a central axis parallelto the direction of z, and a bottom. As the bead storing plate 5 havingsuch plural storing portions 4, a commercially available 384-wellmicrotiter plate can be used, for example. As beads, those having asubstantially equal diameter are prepared simultaneously. The size ofthe beads depends on the maximum diameter or minimum diameter of thecapillary which can be used stably as bead manipulation means. It ispreferred to use spherical beads having a diameter of from about severalμm to several hundred μm, preferably from about 10 μm to 500 μm from theviewpoint of using the beads having a surface area wide enough to fix,to the surface of the bead, the amount of the biomolecular probedetectable from one bead by fluorescent detection. For ease of thefollowing explanation, the external diameter of the bead is assumed tobe 100 μm.

[0049] From a bead container 55, several mg of beads are dispensed by amedicine spoon to each of m×n pieces of the storing portions 4 of thebead storing plate 5. Probes are introduced into the storing portions 4so as to become different in kind by row, or different kinds of probesare introduced into the storing portions, respectively, followed byimmobilization of the probes onto the surfaces of all the beads. By thisoperation, the bead storing plate 5 having plural kinds ofprobe-immobilized bead groups 42 stored therein can be prepared. Theprobe-immobilized bead groups are different in the kind of probes,depending on the position of the storing portions 4. In this embodiment,n kinds of biomolecular probes are prepared and they are immobilizedonto beads stored in storing portions by introducing No. 1 biomolecularprobes in m pieces of storing portions in the first row, No. 2biomolecular probes in m pieces of storing portions in the second row, .. . and No. n biomolecular probes in m pieces of storing portions in then-th row. When the probe immobilized onto bead is a biomolecule which isrelatively chemically stable such as DNA, the bead storing plate 5 canbe stored in a desiccator or a refrigerator. The bead storing plate 5having a solvent such as pure water introduced in each storing portion 4is disposed on a stage for bead storing plate of a bead array chipmanufacturing apparatus which will be described later.

[0050]FIG. 2 is a schematic explanatory view of a bead array chipmanufacturing container, in which FIG. 2A is a planar view of asubstrate constituting a bead array container, while FIG. 2B is anexploded view of the bead array container.

[0051] The bead array container 12 can be manufactured by causing anunprocessed slide glass 11 to stick and adhere to a substrate 10 made ofa quartz slide glass or pyrex slide glass, or made of PDMS on which aplurality of bead disposing flow channels 14 and a capillary passage 20crossing at right angles thereto have been patterned by wet etching.

[0052] The plurality of bead disposing flow channels 14 have across-sectional area permitting passage of only one bead and aredisposed parallel to each other. The bead disposing flow channels 14each has a dam 36 to prevent outflow of beads to the terminal regionwhich is opposite to the capillary passage 20. Between the dam 36 andthe walls of bead disposing flow channel 14, a space is disposed so asnot to block the channel. As will be described later, a liquid isintroduced into the bead disposing flow channel 14 from an openingportion 38 a on the upstream side toward an opening portion 38 b on thedownstream side and a bead is transferred by a liquid stream from theupstream side to the downstream side in the bead disposing flow channel14.

[0053]FIG. 3 is a schematic view of one constitution example of a beadarray chip manufacturing apparatus according to the present invention.This bead array manufacturing apparatus is equipped with a first platemember 1, a second plate member 2 and a third plate member 3.

[0054] On the first plate member 1, disposed via a first electricactuator 8 is a stage 7 which can be equipped thereon with a washingtank 6 and the above-described bead storing plate 5 having, in theplural storing portions 4 thereof, beads conjugated with a probe whichis to bind to a biomolecule such as DNA, RNA or protein. The washingtank 6 contains a cleansing solution suited for preventingcontamination. To heighten the cleansing effect further, an ultrasonictransducer is attached to the washing tank 6. For ease of description, adisposal example of a bead storing plate 65 having 3 storing portions inthe x direction and 5 storing portions in the y direction, 3×5=15storing portions 4 in total is illustrated. The position of the beadstoring plate 5 and the position of the washing tank 6 are controlled bythe first electric actuator 8 movable in the y direction.

[0055] The second plate member 2 has, on the upper portion thereof,cartridge-system container holders 13 a, 13 b for setting the bead arraycontainer 12 therein, a first electromagnetic valve unit 15 forcontrolling the supply of pure water in all the bead disposing flowchannels 14 in the bead array container 12 via the container holder 13a, and a second electromagnetic valve unit 16 necessary for sucking thepure water fed in the bead disposing flow channel 14 via the containerholder 13 b.

[0056] The first electromagnetic valve unit 15 is linked to the waterfeed system 17, while the second electromagnetic valve unit 16 is linkedto a first suction pump 18. In the container holders 13 a, 13 b, achannel is formed which is designed to be connected to the beaddisposing flow channel 14.

[0057] At the lower part of the second plate member 2, disposed is athird electromagnetic valve unit 21 for opening or closing the openingend of the capillary passage 20 in the bead array container 12 throughwhich the bead capturing capillary 19 attached to the third plate member3, which will be described in detail later, is inserted and transferred.

[0058] For ease of explanation, an example of FIG. 3 shows disposal, inthe container holders 13 a, 13 b, of three bead array containers 12arrayed in the x direction. In the practical constitution using a 384(16×24) well microtiter plate as the bead storing plate 5, 16 bead arraycontainers 12 in the form of a slide glass having a size of 25 mm×75 mmand thickness of 3 mm are disposed at center distance of 4.5 mm so thatthe edge of the containers is on the x-y plane and the longitudinal sideruns along the y direction.

[0059] On the third plate member 3, a plurality of bead capturingcapillaries 19 having an inner diameter large enough for sucking onlyone bead in the end of each of the capillaries are arrayed and fixed inone line in the x direction, with the center axes set vertical to theplate member 3. The distance between center axes of the plurality ofbead capturing capillaries 19 is designated as a first predeterminedcenter distance. The third plate member 3 is controlled by a secondelectric actuator 23.

[0060] To suck and retain only one bead in the end portion of each ofthe bead capturing capillaries, it is only necessary to satisfy thefollowing relationships: ID<<R and R≦OD<2R wherein ID represents theinner diameter of the bead capturing capillary 19, R represents a radiusof bead, and OD represents the external diameter of each of the beadcapturing capillaries. Use of a glass or stainless capillary having aninner diameter of 50 μm and an external diameter of 100 or 150 μm issuited when the bead has a radius of 100 μm. One end of the beadcapturing capillary 19 is linked to a second suction pump 24 via afourth electromagnetic valve unit 25.

[0061] In the bead array container 12, the capillary passage 20 which isan induction passage of the bead capturing capillary 19 and the beaddisposing flow channel 14 for disposing beads are formed. The capillarypassage 20 is formed in the z direction of FIG. 3 and the bead disposingflow channel 14 is formed in the y direction of FIG. 3. In the beadarray container 12, the capillary passage 20 and the bead disposing flowchannel 14 cross each other.

[0062] To array a plurality of beads in the bead disposing flow channel14 of the bead array 12 while sucking beads one by one therein andretaining the sucking order of the beads, it is only necessary tosatisfy the following relationship: R<X<((2+{square root}2)/2)R, inwhich X represents each of the two widths of the bead disposing flowchannel 14 when the flow channel is in the form of a quadratic prism.The capillary passage 20, on the other hand, must have a width enoughfor inserting the bead capturing capillary 19 therein so that it isnecessary to satisfy the following relationship: OD<X′, wherein X′represents each of the two widths of the capillary passage 20 when thepassage is in the form of a quadratic prism. For example, it ispreferred to use a bead array container 12 with the bead disposing flowchannel 14 having a width X of 130 μm, and the capillary passage 20having a width X′ of 200 μm.

[0063] For confirmation whether the bead is retained in the end of thebead capturing capillary 19 or not, an image sensor 22 is disposed at aposition sandwiched by the first plate member 1 and the second platemember 2, that is, at an intermediate position of the reciprocatingmovement of the bead capturing capillary 19 between the bead storingplate 5 and the bead array container 12. The number of the lenses forthe image sensor 22 corresponds to the number of bead capturingcapillaries 19 so that a plurality of single beads can be detectedsimultaneously.

[0064] The first electric actuator 8, second electric actuator 23, firstsuction pump 18, second suction pump 24, first electromagnetic valveunit 15, second electromagnetic valve unit 16, third electromagneticunit 21, fourth electromagnetic valve unit 25 and image sensor 22constituting the driving portion of the whole system illustrated in FIG.3 are controlled totally by a computer (controller) 26.

[0065]FIGS. 4A and 4B are schematic views illustrating the way how thebead array container is installed in the bead array chip manufacturingapparatus and it shows an x-y cross-section of the bead array containerloaded on the bead array chip manufacturing apparatus of FIG. 3.

[0066]FIG. 4A illustrates the bead array container 12 illustrated inFIG. 2 installed in the cartridge-system container holders 13 a, 13 b ofthe bead array manufacturing apparatus. The bead array container 12 isinserted in a concave portion of each of the container holders 13 a, 13b. As illustrated in FIG. 4B, then, the container holders 13 a, 13 b arepressed against the bead array container 12 to insert, in the beaddisposing flow channel 14, a PDMS-made socket 34 existing at the end ofthe flow channel 40 in the container holders 13 a, 13 b. This makes itpossible to reliably link the flow channel 40 in the container holder tothe bead disposing flow channel 14 on the bead array container 12. Inthis manner, the desired number of the bead array containers 12 are setin the container holders 13 a, 13 b. The number of the bead arraycontainers is usually the number corresponding to the number m of thestoring portions in one row of the bead storing plate. In other words, mpieces of the containers are loaded.

[0067] A description will next be made of the operation procedure of thebead array chip manufacturing apparatus of the present invention.

[0068]FIG. 5 is a schematic diagram illustrating the y-z cross-sectionof the bead array chip manufacturing apparatus in the preset mode. Byturning on a computer 26, the whole system is in the preset mode and thefirst suction pump 18, second suction pump 24 and water feed system 17start operation, while the first electromagnetic valve unit 15, secondelectromagnetic valve unit 16 and third electromagnetic unit 21 areclosed. By the control of the second electric actuator 23, the thirdplate member 3 goes down and the bead capturing capillary 19 isintroduced into the capillary passage 20 in the bead array container 12.

[0069] On the input screen of the computer 26, the number of the beadarray container 12 and the storing portion 4 of the bead, which has anassigned inspection item, to be disposed in the container are selected.It is needless to say that a driving soft of the computer includes, as adefault program, simple steps such as disposing beads, which are in thestoring portion 4 of the bead storing plate 5, one by one in the beaddisposing flow channel 14 along one direction of y. Clicking on thedrive button of the computer is therefore enough for carrying out suchsimple steps.

[0070]FIG. 6 illustrates one example of the display screen of thecomputer 26. Indicated on the upper side 61 of the display screen is theinformation of the bead storing plate, more specifically, ID 62 of adesignated bead storing plate, a layout 64 indicating the layout of thestoring portions of the bead storing plate with the storing portionnumber 63, and information (probe information) 65 of a bead stored ineach storing portion. The information of the storing portion on the beadstoring plate is linked with that of the bead so that a proper bead tobe arrayed can be selected from the bead information display screen 65(channel in which bead is arrayed is also selected). Information on thebead storing plate previously manufactured can easily be called up fromthe ID of the plate if it is registered in the computer.

[0071] On the lower side 71 of the display screen, manufacturing results(prediction) 73 of the bead array chip reflecting the information of thebead selected on the upper side of the screen are indicated togetherwith the chip ID 72. Details 74 of the bead array chip are alsoindicated on the right side of the screen. The computer also has afunction of showing the ID of the bead storing plate necessary forarraying, if the array results of the bead array chip manufacturedbefore are registered in the computer. The detailed information of thebead array chip shown by the computer include test objects and thenumber of test items (the number of beads arrayed). The data on thescreen can be printed out after the manufacture of the bead array chipso that not only the bead array chip but also its data can be providedto users simultaneously.

[0072] The correspondence between the ID of the bead storing platehaving probe-immobilized beads stored therein in advance and the kind ofthe probe immobilized to the bead stored in each storing portion of thebead storing plate is saved in a memory. In addition, gene tests areclassified by the name of a disease and the kinds of the probe necessaryfor each gene test are also stored in the memory. Then, by selecting theintended disease name from a plurality of disease names displayedselectably on the screen of the computer which controls the bead arraychip manufacturing apparatus, it becomes possible to automatically andconveniently manufacture a bead array chip having probes necessary for agene test of the disease.

[0073] It is preferred to provide users with the bead array chip inwhich probe-immobilized beads have been arrayed in a predeterminedorder, together with a storage medium containing the information on thekind of the probe immobilized to each bead stored in the chip.

[0074] When plural kinds of beads immobilized with a probe such as DNA,RNA or protein necessary for the test of a gene related to a certaindisease, or plural kinds of the beads immobilized with a probe necessaryfor presumption of an exogenous gene of, for example, an infectiousdisease are prepared in advance in respective bead storing plates andstored under a chemically stable state, a bead array chip necessary inthe medical site can be manufactured and provided for test immediately,by taking the bead storing plate from the stored place, installing it onthe bead array chip manufacturing device, and then arraying the beadsimmobilized with probes necessary for the test of patients in the beadarray container.

[0075] As is apparent from FIG. 3, the total number of beads disposed inthe bead disposing flow channel 14 of each bead array container 12depends on the number of the storing portions 4 arrayed along the ydirection of the bead storing plate 5. If one bead storing plate 5cannot cover all the inspection items, a stage 7 permitting loading of aplurality of bead storing plates 5 thereon can be used. Alternatively, amechanism for loading a plurality of bead storing plates in turn can beused.

[0076] After completion of the above-described setting, automaticoperation of the apparatus starts by clicking on a bead disposalstarting button of the computer 26.

[0077]FIGS. 7A to FIG. 71 are schematic cross-sectional viewsillustrating steps of taking out one bead 28 from a plurality of beads28 stored in a certain storing portion 4 and introducing it in the beaddisposing flow channel 14. The bead array container illustrated in thesedrawings are each formed of one capillary passage 20 and three beaddisposing flow channels 14.

[0078]FIG. 7A illustrates the state after the stage 7 is transferred sothat an opening of the storing portion 4 having the intended beadtherein comes opposite to an opening of the bead capturing capillary 19in the z direction by the control of the first electric actuator 8. Thethird electromagnetic valve unit 21 disposed below the second platemember 2 is opened. By the second electric actuator 23, the third platemember 3 is transferred in the z direction to project the end of thebead capturing capillary 19 from the lower end of the capillary passage20 of the bead array container 12. This diagram illustrates immediatelybefore the bead capturing capillary 19 is inserted into the intendedstoring portion 4.

[0079] As illustrated in FIG. 7B, the third plate member 3 movesdownward in the z direction by the control of the second electricactuator 23 and the lower end of the bead capturing capillary 19 isinserted inside of the storing portion 4. At this time, the fourthelectromagnetic valve unit 25 is opened to cause suction in the beadcapturing capillary 19. In the procedure of FIG. 7B, the bead 28 iscaptured in the end of the bead capturing capillary 19. A plurality ofbeads 29 adsorb to not only the end of the bead capturing capillary 19but also to its outside wall by electrostatic attraction.

[0080]FIGS. 7C to 7F illustrate a step of moving the bead capturingcapillary 19 attached to the third plate member 3 in the z direction andpulling it back to the inside of the bead array container 12 by thecontrol of the second electric actuator 23. FIG. 7C illustrates the beadcapturing capillary 19 existing in pure water in the storing portion 4,leaving the beads 28 suspended on the bottom of the storing portion 4.At this time, a plurality of beads 28 adsorb to the opening portion andoutside wall of the bead capturing capillary 19. FIG. 7D illustrates thebead capturing capillary 19 moved in the z direction more from that ofFIG. 7C and the end portion of the bead capturing capillary 19 is justpassing through a gas-liquid interface 29. A plurality of extra beads 28which have adsorbed to the outer surface of the bead capturing capillary19 are scaled off below the surface of water by the surface tension ofthe gas-liquid interface 29.

[0081]FIG. 7E illustrates the end of the bead capturing capillary 19taken out completely from pure water of the storing portion 4 into theair. At this time, the bead capturing capillary 19 has, at an endthereof, only one bead 28. In the air, at an intermediate position ofthe transfer of the end of the bead capturing capillary 19 from thestoring portion 4 to the capillary passage 20 in the bead arraycontainer 12, the image of the bead 38 in the end of the bead capturingcapillary 19 is detected by the image sensor 22.

[0082]FIG. 8 is a schematic diagram for explaining an image detectionmethod by the image sensor 22. FIG. 8A illustrates an example ofconstitution of an image detection system, while FIG. 8B illustrates thebead detected on the tonal range setting screen of a light and a sensorscreen.

[0083] Image detection of a scattered light from the bead 28 may beperformed by using illumination 30 with a white light emitting diode 56and focusing on the bead 28 (FIG. 8A). By setting the program of theimage sensor 22 in advance so as to cut the color gradation level 57 ofa background light and leave only the color gradation level 32 of thescattered light of the bead, the scattered light emitted from one bead28 can be detected on a sensor screen 33 in the form substantiallyreflecting the form of the bead 28 when the bead 28 is retained in theend of the bead capturing capillary 19 (FIG. 8B). When the bead 28 isnot retained in the end of the bead capturing capillary 19, on the otherhand, scattered light of a predetermined gradation level cannot bedetected on the sensor screen 33. If the bead 28 is not detected by theimage sensor 22, all the above-described steps FIGS. 7A to 7E arerepeated again.

[0084] After the bead 28 is detected by the image sensor 22, the end ofthe bead capturing capillary 19 is transferred to the intersection ofthe capillary passage 20 and the bead disposing flow channel 14 in thebead array container by the control of the second electric actuator 23,as illustrated in FIG. 7F. FIG. 7F illustrates the bead capturingcapillary 19 located at the second intersection of the three beaddisposing flow channels 14 in the bead array container 12, counted inthe z direction from the second plate member 2.

[0085]FIG. 7G and FIG. 7H are each a y-z cross-sectional viewillustrating a step of introducing the bead 28 retained in the beadcapturing capillary 19 into the bead disposing flow channel 14.

[0086] As illustrated in FIG. 7G, the fourth electromagnetic valve unit25 is closed, whereby the suction in the bead capturing capillary 19 atthe above-described intersection 34 by the second suction pump 24 isterminated. At this time, the bead 28 in the end of the bead capturingcapillary 19 is still retained in the end under the vacuum conditioninside of the capillary 19. Simultaneously with the control of thefourth electromagnetic valve unit 25, the third electromagnetic valveunit 21 is also closed.

[0087] Then, the first electromagnetic valve unit 15 is opened and purewater is fed from one end of the bead disposing flow channel 14 throughthe water feed system 17. At this time, the second electromagnetic valveunit 16 linked to another end of the bead disposing flow channel 14 isalso opened and the pure water fed by the water feed system is thensucked by the first suction pump 18. By making use of a water stream 35generated by the supply of the pure water and suction of the pure waterthus fed, the bead 28 is physically scaled off from the end of the beadcapturing capillary 19 and charged in the bead disposing flow channel 14by this water stream 35.

[0088] In the bead disposing flow channel 14 of the bead array container12, a dam 36 is disposed. The first bead introduced in the flow channelis disposed as illustrated in FIG. 7H, blocked by the dam 36.

[0089] According to the above description, FIG. 7F illustrates the beadcapturing capillary 19 located at the second intersection of the threebead disposing flow channels 14 in the bead array container 12, countedin the z direction from the second plate member 2. In practice, a beadgroup having a desired sequence can be formed in any bead disposing flowchannel 14 by the control of the third plate member 3 in the zdirection. As a result, a bead array chip having two-dimensionallyarrayed beads on a y-z plane is manufactured. In this Embodiment, onlythe results of the bead 28 arrayed in the second bead disposing flowchannel 14 are described for brevity.

[0090]FIG. 7I illustrates the confirmation of the presence or absence ofthe bead 28 in end of the bead capturing capillary 19 by the imagesensor 22 after completion of the introduction of the bead 28. When nobead 28 exists in the end of the bead capturing capillary 19 as a resultof the image detection in FIG. 71, the disposing step of one bead in thebead disposing flow channel 14 is completed. When the image sensor 22judges that the bead 28 exists, on the other hand, steps illustrated inFIGS. 7F to 7I are repeated again.

[0091] The beads 28 contained in the storing portions 4 are immobilizedwith probes different in kind, depending on the line of the storingportions arrayed in the y direction of FIG. 3. By controlling the firstelectric actuator 8 to move the first plate member 1 by a desireddistance in the y direction and successively change the storing portion4 which can be selected freely by the movement in the y direction, andrepeating the above-described operations as illustrated in FIGS. 7A toFIG. 7I, the beads immobilized with different probes respectively can bedisposed in the bead disposing flow channel 14 in the bead arraycontainer 12 in the desired order.

[0092]FIG. 7J and FIG. 7K each illustrates the washing step of the endof the bead capturing capillary 19 in the washing tank 6. After one bead28 is introduced into the bead disposing flow channel 14 but beforeanother bead 18 is taken out from the storing portion 4, the end portionof the bead capturing capillary 19 is dipped in a detergent in thewashing tank 6 and washed therewith.

[0093] The steps as described above in FIG. 7A to FIG. 7K arefundamental for disposing one bead 28. Whenever the bead 28 isintroduced in the flow channel based on the information on anclassification index discriminated by the storing position of the beadstoring plate 5 and stored in a computer in advance, the index and thenumber of beads 28 introduced in the bead disposing flow channel 14 inthe bead array container 12 are recorded sequentially.

[0094]FIG. 9 is a flow chart of the manufacturing process of the beadarray chip described above.

[0095] The manufacturing process of the bead chip array according to thepresent invention can be classified in two stages, that is, pretreatmentand manufacture of a bead array chip. In the pretreatment, asillustrated in FIG. 1, minute beads are dispensed in each storingportion 4 of the bead storing plate 5 (S11) and biomolecular probe suchas DNA probe is introduced in each storing portion 4 to immobilize it onthe surface of the bead (S12). Then, the information on the bead storingplate and the bead (probe kind) in each storing portion are input in acomputer (S13). A plurality of bead storing plates equipped with storingportions containing beads immobilized with biomolecular probes differentin kind are prepared and they can be registered after ID numbering.Then, a desired bead storing plate 5 is disposed at a predeterminedposition on the stage 7 of the bead array chip manufacturing apparatus(S14) and the required number of bead array containers 12 are installedin the container holders 13 a, 13 b of the bead array chip manufacturingapparatus (S15).

[0096] In the next place, a bead array chip is manufactured using thebead array chip manufacturing apparatus.

[0097] Based on the registered plate information and bead information,beads to be arrayed in a container, their arraying order, and desiredflow channel in a bead array container are selected on the displayscreen of the computer (controller) (S16) and an arraying start buttonis clicked (S17). Then, the bead array chip manufacturing apparatusautomatically manufactures a bead array chip as designed. Describedspecifically, the bead capturing capillary 19 is moved downward to dipthe end thereof in pure water in the intended storing portion 4 of thebead storing plate 5. After one bead is captured (S18), the capillary ismoved upward. During this procedure, capture of one bead in the end ofthe bead capturing capillary 19 is confirmed by image detection (S19).After confirmation that the bead has been captured in the bead capturingcapillary 19, the bead capturing capillary 19 is moved upward further todraw the bead in the bead array container. The bead is located at apredetermined bead disposing channel and then, introduced in the beaddisposing channel by a water stream (S20). The bead capturing capillary19 which has lost the bead from its end is moved downward below the beadarray container in order to capture a next bead. During this descent, itis confirmed by image detection that the capillary has no bead in itsend (S21). The end of the bead capturing capillary 19 is inserted andwashed in the washing tank 6 (S22). The operations of from step 18 tostep 22 are repeated the number of times corresponding to the number ofbeads, whereby the beads arraying operation is completed (S23). Upon useof the bead array chip thus manufactured, information (kinds of beads,arraying order) on the bead array chip is obtained from the computer andbased on the information, a bead array chip is selected or test resultsare analyzed.

[0098]FIG. 10A and FIG. 10B are each an explanatory view illustrating anattaching method of a sample injection jig 44 to the bead array chipthus manufactured. After completion of the disposal of the beads 28, thebead array container 12 is removed from the container holders 13 a, 13b, two opening portions 37 of the capillary passage are sealed with aPDMS-made exclusive cap 41. In the opening portion 38 of the beaddisposing flow channel, the sample injection jig 44 for introducing atest sample is inserted.

[0099] The sample injection jig 44 is equipped with a sample injectioncapillary 45 having an inner diameter smaller than the external diameterof the bead. As illustrated in FIG. 10B, the end of the sample injectioncapillary 45 is designed so that it exists downstream of theintersection between the capillary passage 20 and the bead disposingflow channel 14. A joint of the sample injection jig 44 with the beaddisposing flow channel is made of a PDMS material and it is adhered andfixed safely without causing leakage. FIG. 10B is also a diagram of theform employed when the bead array chip manufactured by the bead arraychip manufacturing apparatus of the present invention is marketed as aninexpensive genetic testing tool. Users purchase the bead array chip,together with a storage medium including its specific data on the kindsof the probes immobilized on the beads in the chip and arraying order ofthe beads.

[0100] A description will next be made of a genetic testing method usingthe bead array chip thus manufactured. An example which will bedescribed below is that of the use of a bead array chip having a DNAprobe array, which has a desired sequence and is formed in the beaddisposing flow channel 14, for the hybridization of a specificfluorescent-labeled target DNA on the DNA probe array.

[0101] In this Embodiment, 24 kinds of 18mer synthetic oligonucleotideswhich are different in the base sequence and have been modified with a5′-thiol group were used as the probe DNA. In addition to the 24 kindsof the probe DNAs, another two kinds are prepared. They are a singlestranded target DNA 49 having a Cy3-labeled sequence 3 and asingle-strand target DNA 50 having a TexasRed-labeled sequence 4complementary to the single-strand DNA probe-immobilized bead 47 whichhas been stored as a fifth bead in the bead disposing flow channel 14and has Sequence 1 and a single-strand DNA probe immobilized bead 48which has been stored as a tenth bead and has Sequence 2, respectively.(Sequence 1) 5′-thiol-ATCT....CCTC (Sequence 2) 5′-thiol-CTAC....GACG(Sequence 3) 5′-Cy3-GAGG....AGAT (Sequence 4) 5′-TexasRed-CGTC....GTAG

[0102] A preparation process of the bead 28 conducted prior to thedisposal of the bead in the bead array container 12 will next bedescribed. In this Embodiment, amino-modified glass beads having anexternal diameter of 100 μm were used and they were prepared inaccordance with the schematic view illustrated in FIG. 1. A plurality ofbeads are reacted in a 0.01% N-(4-maleimidobutyryloxy)succinimidesolution (ethanol: 50%, dimethylsulfoxide: 50%) of room temperature for1 hour, followed by washing with a mixture of 50% ethanol and 50%dimethylsulfoxide to prepare maleimide-modified beads. Then, 24 kinds ofprobe DNAs are prepared. Beads immobilized with the same kind of probeDNA are made for 16 storing portions arrayed in a row in the x directionand beads immobilized with different kinds of probe DNAs are made forstoring portions arrayed in the y direction. The probe DNA isimmobilized to the maleimide-modified beads by reacting with syntheticDNA which has a different base sequence and has been modified with5′-end thiol group. After reaction in a 0.11 nM synthetic DNA-20 mMphosphate buffer (pH 7.0) of room temperature for 1 hour, the reactionmixture is washed successively with a 20 mM phosphate buffer (pH 7.0)solution and water to yield a DNA probe immobilized bead group 42.

[0103] The bead storing plate having the thus-prepared beads storedtherein is set on a stage of the apparatus as illustrated in FIG. 3. Bythe above-described procedure, beads 28 having 24 kinds of test probeswhich differ with each row of the storing portions arrayed in the ydirection of a 384-well microtiter plate are introduced one by one inthe bead disposing flow channel 14 in the bead array container 12,whereby a bead array is manufactured.

[0104]FIG. 11 is a schematic view illustrating a method of hybridizationtest by using the bead array chip of the present invention in whichDNA-immobilized beads have been arrayed. It illustrates the bead arraychip 52 made in this Embodiment and 24 different kinds of beads arearrayed in one bead disposing flow channel 14. In another two beaddisposing flow channels 14 of one bead array chip 52, the same beadgroup is formed. In the cross-sectional view of FIG. 11, only one beaddisposing channel 14 is illustrated among three bead disposing flowchannels 14.

[0105] An example of a hybridization reaction at 45° C. by pouring a 20mM phosphate buffer (pH 7.0) solution containing a single strand targetDNA 49 and a single strand target DNA 50, each in an amount of 1 μM, inthe bead disposing flow channel 14 of the bead array chip 52 of FIG. 11will next be described. The solution was fed to the bead disposing flowchannel 14 through a syringe pump or the like. After completion of thereaction, a remaining target DNA which had not contributed to thehybridization reaction was washed successively with a 20 mM phosphatebuffer (pH 7.0) solution and pure water, and then dried. With a mercurylamp as a light source, fluorescence microscopic observation of eachbead in the capillary of the bead array was performed by successivelyusing a long pass filter for Cy3 and a long pass filter for TexasRedmainly for the emission wavelength of Cy3 an TexasRed, respectively.

[0106]FIGS. 12A to 12C illustrate the results of the hybridizationreaction under the above-described conditions by using a bead arraychip. FIG. 12A is a transmission microscopic image of the bead arraychip 52, FIG. 12B illustrates the fluorescence microscopic observationresults through the long pass filter for Cy3, and FIG. 12C illustratesthe fluorescence microscopic observation results through the long passfilter for TexasRed.

[0107] As is apparent from FIG. 12B and FIG. 12C, among the 24 beads 28thus arrayed, the 5-th bead and 10-th bead emit fluorescence 53 of Cy3and fluorescence 54 of TexasRed, respectively. This suggests that thehybridization has occurred between the single strand target DNA 49 andthe single strand DNA probe immobilized bead 47 and between the singlestrand target DNA 50 and the single strand DNA probe immobilized bead48. By the apparatus in this Embodiment, it is possible to manufacture aDNA probe array in the bead array container 12 by a desired orderwithout having an influence on the probe.

[0108] According to the present invention, a bead array chip havingbiomolecule-immobilized beads arrayed therein can be manufacturedefficiently at a low manufacturing cost.

What is claimed is:
 1. A bead array chip manufacturing apparatus, whichcomprises: a stage for supporting a bead storing plate having aplurality of bead storing portions, a stage driving portion for drivingthe stage, a container retaining portion for retaining a containerhaving a plurality of first channels disposed substantially in parallelwith each other and a second channel crossing therewith, a capillarywhich is movable and passes through the second channel of the container,a capillary driving portion for vertically moving the capillary, asuction unit connected to the upper end of the capillary for generatinga suction force at the lower end of the capillary, a fluid circulatingunit for generating a uniflow of a fluid in the first channels of thecontainer retained by the container retaining portion, and a controllingportion for controlling the stage driving portion, capillary drivingportion, suction unit and fluid circulating unit; and manufactures abead array chip in which beads stored respectively in the plurality ofstoring portions of the bead storing plate are arrayed in apredetermined order in the first channel of the container.
 2. A beadarray chip manufacturing apparatus of claim 1, wherein the capillarydriving portion drives a plurality of capillaries simultaneously and thecontainer retaining portion retains the number of the containers equalto those of the capillaries.
 3. A bead array chip manufacturingapparatus of claim 1, wherein the capillary has an inner diametersmaller than the diameter of the bead and an external diameter greaterthan the diameter of the bead but not greater than twice as much as thediameter of the bead.
 4. A bead array chip manufacturing apparatus ofclaim 1, wherein the container retaining portion further comprises afirst retaining member having a flow channel linked to one end of thefirst channel and retaining one end portion of the container, and asecond retaining member having a flow channel linked to the other end ofthe first channel and retaining the other end portion of the container;and the fluid circulating unit further comprises a fluid injection unitconnected to the first retaining member and a fluid suction unitconnected to the second retaining member.
 5. A bead array chipmanufacturing apparatus of claim 1, wherein the bead has a diameterranging from 10 μm to 500 μm and has a surface immobilized with abiomolecular probe.
 6. A bead array chip manufacturing process formanufacturing a bead array chip having plural kinds of beads arrayed ina predetermined order in, in a container having a plurality of firstchannels disposed substantially in parallel with each other and a secondchannel crossing the plurality of first channels, each of the firstchannels, which comprises: lowering a capillary inserted in the secondchannel and sucking and retaining one bead in one end of the capillary,lifting the capillary to position the beads retained in the one end ofthe capillary in the desired channel of the plurality of first channels,terminating the suction of the capillary, and generating a uniflow of afluid in the second channel.
 7. A bead array chip manufacturing processof claim 6, further comprising disposing a plurality of the containersand simultaneously driving a plurality of the capillaries each insertedin the second channel of the containers.
 8. A bead array chipmanufacturing process of claim 6, wherein the bead has a surfaceimmobilized with a biomolecular probe.